We propose to create models of beta-thalassemia by altering beta-globin genes in murine ES cells via homologous recombination (HR). Using various HR strategies for gene replacement, we will substitute a "marked" murine beta-globin gene for the normal beta-globin gene in ES cells. By analyzing the effects of the various HR techniques on expression of the marked globin gene, we will determine the optimal HR strategy for the maintenance of normal levels of expression and developmental control. We will then replace the mouse beta-globin genes with normal and thalassemic human beta- globin genes. The expression of the human beta-globin genes will be determined in ES cells differentiated into erythroid cells in vitro and during hematopoiesis in reconstituted mice. These experiments will establish the feasibility of recapitulating normal and thalassemic human beta-globin gene expression after homologous recombination in the murine background. We will also test the feasibility of the homologous recombination approach for correcting thalassemia by replacing a thalassemic mouse beta-globin gene in ES cells and determining the consequences on globin expression and chain balance in vitro and in reconstituted mice. In another series of experiments, we will introduce a human chromosome 11 carrying a thalassemic beta-globin gene into ES and MEL cells by microcell fusion. The thalassemic gene will then be converted into a normal human beta-globin gene via homologous recombination, and the consequences on expression determined. This approach will generate homologous recombination cassettes and strategies that can be used in the future to correct such defects in human cells. In addition, we will replace the entire mouse beta-globin gene locus in ES cells with the entire human beta-globin gene domain, including distal regulatory elements. Using this "locus replacement" strategy, we will create mice that are homozygous for the human beta-globin locus containing either the normal or thalassemic beta-globin gene, as well as normal/thalassemic heterozygotes. This strategy will provide in vivo models for the human beta-thalassemias, as well as for the regulation of normal beta-globin gene expression.